Systems biology is devoted to comprehensive studies of biological components with interrelated mechanisms across resolution scales over six orders of magnitude, involving molecules, sub-cellular features, cells, organisms, and entire species. Living systems are highly complicated, dynamic, and often unpredictable. To understand and manipulate these systems, quantitative measurements of interacting components and clusters are necessary using systematic and microscopic technologies such as microscopies, genomics, proteomics, bioinformatics, in vivo or in situ imaging, and computational models. Regenerative medicine utilizes principles of biology and engineering to develop and transplant engineered substitute tissues and organs, with various protocols for cell seeding onto porous scaffolds during incubation. These constructs are then expected to restore or regenerate functionality of diseased tissues or organs. Engineered tissue growths are rather sophisticated, and as natural biological counterparts, they usually recapitulate normal developmental processes. Hence, systematic and microscopic technologies are critical for evaluating engineered tissue prior and post implementation.
Microscopy is the principal observational tool and has made important contributions to the understanding of biological systems and engineered tissues. However, imaging depth of optical microscopy has been fundamentally limited to millimeter or sub-millimeter due to multiple scattering of light in a biological sample. Conventional microscopy techniques utilize visible light or electron sources. Optical microscopy can be divided into transmission (i.e., wide-field microscopies for snap-shot of 2D images in terms of light absorption, phase contrast, or dark-field signals) and emission modes (i.e., wide-field fluorescence microscopy, confocal laser scanning microscopy, and two-photon fluorescence microscopy).
These microscopic modalities can be used for in vitro and in vivo studies of cultured cell/tissue samples or small animals. Image resolution of optical microscopy is diffraction limited to about 200 nm with single objective techniques and about 120 nm with confocal techniques. With appropriate sample preparation, stochastic information and innovative interference techniques, about 100 nm resolution may be achievable. Three-dimensional image cubes can be obtained with optical sectioning of about 200 nm lateral resolution and about 500 nm axial resolution.
Ultimately, multiple scattering prevents these techniques from imaging thick samples. Photoacoustic tomography permits scalable resolution at imaging depths up to about 7 cm with a depth-to-resolution ratio about 200. Photoacoustic microscopy aims at millimeter imaging depth, micron-scale resolution and absorption contrast, which could be used to characterize the structure of the scaffold but is generally not as sensitive and specific as fluorescence and bioluminescence imaging.